System and method for temperature referencing for melt curve data collection

ABSTRACT

The present invention relates to systems and methods of temperature referencing for melt curve data collection. More specifically, the present invention relates to systems and methods for collecting DNA melt curve data for a DNA sample and a temperature reference material.

CROSS REFERENCE OF RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.12/144,307, filed on Jun. 23, 2008, which is incorporated herein byreference in its entirety.

BACKGROUND

Field of the Invention

The present invention relates to systems and methods of temperaturereferencing for melt curve data collection. More specifically, thepresent invention relates to systems and methods for collecting DNA meltcurve data for a DNA sample and a temperature reference material.

Description of Related Art

The detection of nucleic acids is central to medicine, forensic science,industrial processing, crop and animal breeding, and many other fields.The ability to detect disease conditions (e.g., cancer), infectiousorganisms (e.g., HIV), genetic lineage, genetic markers, and the like,is ubiquitous technology for disease diagnosis and prognosis, markerassisted selection, correct identification of crime scene features, theability to propagate industrial organisms and many other techniques.Determination of the integrity of a nucleic acid of interest can berelevant to the pathology of an infection or cancer. One of the mostpowerful and basic technologies to detect small quantities of nucleicacids is to replicate some or all of a nucleic acid sequence many times,and then analyze the amplification products. PCR is perhaps the mostwell-known of a number of different amplification techniques.

PCR is a powerful technique for amplifying short sections of DNA. WithPCR, one can quickly produce millions of copies of DNA starting from asingle template DNA molecule. PCR includes a three phase temperaturecycle of denaturation of DNA into single strands, annealing of primersto the denatured strands, and extension of the primers by a thermostableDNA polymerase enzyme. This cycle is repeated so that there are enoughcopies to be detected and analyzed. In principle, each cycle of PCRcould double the number of copies. In practice, the multiplicationachieved after each cycle is always less than 2. Furthermore, as PCRcycling continues, the buildup of amplified DNA products eventuallyceases as the concentrations of required reactants diminish. For generaldetails concerning PCR, see Sambrook and Russell, Molecular Cloning—ALaboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology,F. M. Ausubel et al., eds., Current Protocols, a joint venture betweenGreene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(supplemented through 2005) and PCR Protocols A Guide to Methods andApplications, M. A. Innis et al., eds., Academic Press Inc. San Diego,Calif. (1990).

Real-time PCR refers to a growing set of techniques in which onemeasures the buildup of amplified DNA products as the reactionprogresses, typically once per PCR cycle. Monitoring the accumulation ofproducts over time allows one to determine the efficiency of thereaction, as well as to estimate the initial concentration of DNAtemplate molecules. For general details concerning real-time PCR seeReal-Time PCR: An Essential Guide, K. Edwards et al., eds., HorizonBioscience, Norwich, U.K. (2004).

More recently, a number of high throughput approaches to performing PCRand other amplification reactions have been developed, e.g., involvingamplification reactions in microfluidic devices, as well as methods fordetecting and analyzing amplified nucleic acids in or on the devices.Thermal cycling of the sample for amplification is usually accomplishedin one of two methods. In the first method, the sample solution isloaded into the device and the temperature is cycled in time, much likea conventional PCR instrument. In the second method, the sample solutionis pumped continuously through spatially varying temperature zones. See,for example, Lagally et al. (Anal Chem 73:565-570 (2001)), Kopp et al.(Science 280:1046-1048 (1998)), Park et al. (Anal Chem 75:6029-6033(2003)), Hahn et al. (WO 2005/075683), Enzelberger et al. (U.S. Pat. No.6,960,437) and Knapp et al. (U.S. Patent Application Publication No.2005/0042639).

Microfluidic systems are systems that have at least one channel throughwhich a fluid may flow, which channel has at least one internalcross-sectional dimension, (e.g., depth, width, length, diameter) thatis less than about 1000 micrometers. Typically, microchannels have across-sectional dimension of about 5 microns to about 500 microns and adepth of about 1 micron to about 100 microns.

Melt curve analysis is an important technique for analyzing nucleicacids. In this method, a double stranded nucleic acid is denatured inthe presence of a dye that indicates whether the two strands are boundor not. Examples of such indicator dyes include non-specific bindingdyes such as SYBR® Green I, whose fluorescence efficiency dependsstrongly on whether it is bound to double stranded DNA. As thetemperature of the mixture is raised, a reduction in fluorescence fromthe dye indicates that the nucleic acid molecule has melted, i.e.,unzipped, partially or completely. Thus, by measuring the dyefluorescence as a function of temperature, information is gainedregarding the length of the duplex, the GC content or even the exactsequence. See, for example, Ririe et al. (Anal Biochem 245:154-160,1997), Wittwer et al. (Clin Chem 49:853-860, 2003), Liew et al. (ClinChem 50:1156-1164 (2004), Herrmann et al. (Clin Chem 52:494-503, 2006),Knapp et al. (U.S. Patent Application Publication No. 2002/0197630),Wittwer et al. (U.S. Patent Application Publication No. 2005/0233335),Wittwer et al. (U.S. Patent Application Publication No. 2006/0019253)and Sundberg et al. (U.S. Patent Application Publication No.2007/0026421).

A number of commercial instruments exist that perform thermal melts onDNA. Examples of available instruments include the Idaho Technology HR-1high resolution melter and the Idaho Technology LightScanner highresolution melter. The HR-1 high resolution melter has the highestresolution fluorescent signal to noise ratio and temperature resolutionof any commercial device. However, it suffers from a limitation that itcan only analyze one sample at a time, and the sample container must bereplaced manually. Replacement of the container for each test perhapscontributes to run-to-run temperature variability. The LightScanner highresolution melter also has good signal and temperature resolution, andoperates on a 96-well plate sample container. However, it suffers fromsignificant (˜0.3° C.) temperature gradients across the entire plate, asdo other systems based on standard well plates. A typical mode ofoperation for these analyzers is to apply heat to the sample(s) in acontrolled manner to achieve a linear rise in temperature versus time.Simultaneously, a stable continuous fluorescence excitation light isapplied, and emitted fluorescence is collected continuously over fixedintegration time intervals. The fluorescence intensity data is convertedfrom a time basis to a temperature basis based on the knowledge of thetemperature ramp versus time.

One of the difficulties inherent to this method is that the temperaturecontrol system has limited precision and accuracy. For example, thefeedback signal used to control the heater may come from a temperaturesensor that is physically displaced from the sample. During atemperature ramp, heat diffuses from the heat source to the samplethrough the sample container, and hence temperature gradients existwithin the sample and across the sample container as well. A temperaturesensor outside the sample container, even if perfectly accurate, willreport a temperature that is offset somewhat from the instantaneoussample temperature. Furthermore, this offset will depend upon the ramprate, the geometry and the quality of thermal contact between the heaterand the sample container.

There is current market interest in further developing microfluidicgenomic sample analysis systems for detecting and analyzing DNAsequences. The development of these microfluidic systems often entailthe various combinations of channel configurations, inlets, outlets,buffer insertion methods, boluses of genomic sample insertion methods,temperature cycling and control methods, and optical analysis methods.There is also further interest in developing systems and methods fortemperature referencing for melt curve data collection.

Microfluidic melting curve analysis is typically carried out either in astopped flow format or in a continuous flow format. In a stopped flowformat, flow is stopped within a microchannel of a microfluidic devicewhile the temperature in that channel is ramped through a range oftemperatures required to generate the desired melt curve. A drawback tothe stopped flow format is that it does not integrate well in systemswith other flow through processes which require the flow to continuewithout any stoppage. When fluorescent indicator dyes are used tomonitor denaturation, another drawback to the stopped flow format is theloss of fluorescent signal due to dye photobleaching while the thermalramp is being performed.

In a continuous flow format, a melting curve analysis is performed byapplying a temperature gradient along the length (direction of flow) ofa microchannel in a microfluidic device. If the melting curve analysisrequires that the molecules being analyzed be subjected to a range oftemperatures extending from a first temperature to a second temperature,the temperature at one end of the microchannel is controlled to thefirst temperature, and the temperature at the other end of the length iscontrolled to the second temperature, thus creating a continuoustemperature gradient spanning the temperature range between the firstand second selected temperatures. A drawback to certain implementationsof the continuous flow format is that thermal properties of themolecules in the stream must be measured at multiple points along thetemperature gradient to generate the desired melting curve. This makesmeasurement of thermal properties of the molecules in the stream morecomplex than in the stopped flow format, where thermal properties of themolecules in the stream can be measured at a single point to generatethe desired melting curve.

Sundberg et al. (U.S. Patent Application Publication No. 2007/0026421)and Knight et al. (U.S. Patent Application Publication No.2007/0231799), each incorporated by reference herein, describe methods,systems, kits and devices for conducting binding assays using molecularmelt curves in microfluidic devices. Molecule(s) to be assayed can beflowed through microchannels in the devices where the molecule(s)optionally are exposed to additional molecules constituting, e.g.,fluorescence indicator molecules and/or binding partners of the moleculebeing assayed. The molecules involved are then heated (and/or cooled)and a detectable property of the molecules is measured over a range oftemperatures. From the resulting data, a thermal property curve(s) isconstructed which allows determination and quantification of the bindingaffinity of the molecules involved.

Known systems and methods for melt curve analysis suffer from someamount of uncertainty and lack of reproducibility, inter alia, in termsof temperature control and measurement systems. Accordingly, there is acontinuing need to improve the usefulness of the melt curve analysistechnique by reducing the impact of spatial and temporal temperaturefluctuations.

SUMMARY OF THE INVENTION

The present invention relates to systems and methods of temperaturereferencing for melt curve data collection. More specifically, thepresent invention relates to systems and methods for collecting DNA meltcurve data for a DNA sample and a temperature reference material.

Thus, in a first aspect, the present invention provides a system forperforming melt curve data collection comprising a container, a heatingsystem and a detection system, wherein the container comprises at leasttwo chambers that are in close thermal connection and wherein at leastone chamber contains a DNA sample to be tested and at least one chambercontains a temperature reference material. In some embodiments, theheating system supplies heat to all of the chambers simultaneously. Insome embodiments, the chambers contain at least two spatially separatedtemperature reference materials to determine a spatial temperaturegradient. In other embodiments, the temperature reference materialsbracket the DNA sample in both space and time. In further embodiments,the temperature reference material is mixed with the DNA sample. In someembodiments, the detection system is an optical detection system. Inother embodiments, detectable signal from the temperature referencematerial provides feedback to the heating system. In furtherembodiments, the container is a microfluidic chip and the chambers aremicrofluidic channels in the chip. In some embodiments, the containercomprises at least three chambers wherein at least two of the chambersare spatially separated from each other and contain a temperaturereference material to determine a spatial temperature gradient. In otherembodiments, at least two chambers containing DNA samples are locatedbetween two chambers containing the temperature reference material. Insome embodiments, the temperature reference material is a known DNAmixture. In other embodiments, the temperature reference material is athermochromatic material.

In a second aspect, the present invention provides a system forperforming melt curve data collection comprising a container, a heatingsystem and a detection system, wherein the container comprises at leastone chamber that is refillable without moving the container, wherein thecontainer contains a DNA sample to be tested and a temperature referencematerial. In some embodiments, the heating system supplies heat to allof the chambers simultaneously. In some embodiments, the detectionsystem is an optical detection system. In other embodiments, detectablesignal from the temperature reference material provides feedback to theheating system. In some embodiments, the DNA sample and the temperaturereference material are alternated in the chamber. In other embodiments,the temperature reference material brackets the DNA sample in both spaceand time. In further embodiments, the DNA sample and the temperaturereference material are mixed, wherein the temperature reference materialhas a detection signature that is discernible from that of the DNAsample and wherein data from both the sample and the temperaturereference material are collected at the same place and the same time. Insome embodiments, the temperature reference material is also a flowmarker.

In some embodiments, the container is a microfluidic chip and thechambers are microfluidic channels in the chip. In some embodiments, thecontainer comprises at least three chambers wherein at least two of thechambers are spatially separated from each other and contain atemperature reference material to determine a spatial temperaturegradient. In other embodiments, at least two chambers containing DNAsamples are located between two chambers containing the temperaturereference material. In some embodiments, the temperature referencematerial is a known DNA mixture. In other embodiments, the temperaturereference material is a thermochromatic material.

In a third aspect, the present invention provides a method of performingmelt curve data collection comprising providing a container whichincludes at least two chambers that are in close thermal connection,introducing a DNA sample to be tested into at least one of the chambersand a temperature reference material into at least one of the otherchambers, heating the chambers from a first temperature to a secondtemperature, and measuring a detectable property emanating from the DNAsample and a detectable property emanating from the temperaturereference material, wherein the detectable property of the DNA sampleindicates an extent of denaturation of the DNA in the sample and thedetectable property of the temperature reference material is correlatedto the temperature. In some embodiments, the chambers contain at leasttwo spatially separated temperature reference materials and thedetectable property emanating from the temperature reference materialsis measured to determine a spatial temperature gradient. In otherembodiments, the temperature reference materials bracket the DNA samplein both space and time. In some embodiments, the detectable propertyemanating from the temperature reference materials bracketing the DNAsample is measured to determine a spatial temperature gradient and anytemporal fluctuation. In further embodiments, the temperature referencematerial is mixed with the DNA sample.

In some embodiments, the detectable property is measured with an opticaldetection system. In other embodiments, detectable signal from thetemperature reference material provides feedback to the heating system.In further embodiments, the container is a microfluidic chip and thechambers are microfluidic channels in the chip. In some embodiments, thecontainer comprises at least three chambers wherein at least two of thechambers are spatially separated from each other and contain temperaturereference materials and the detectable property emanating from thetemperature reference materials is measured to determine a spatialtemperature gradient. In other embodiments, at least two chamberscontaining DNA samples are located between two chambers containing thetemperature reference material. In some embodiments, the temperaturereference material is a known DNA mixture. In other embodiments, thetemperature reference material is a thermochromatic material.

In a fourth aspect, the present invention provides a method ofperforming melt curve data collection comprising providing a containerwhich includes at least one chamber that is refillable without movingthe container, introducing a DNA sample to be tested into at least oneof the chambers and a temperature reference material into at least oneof the chambers, heating the chambers from a first temperature to asecond temperature, and measuring a detectable property emanating fromthe DNA sample and a detectable property emanating from the temperaturereference material, wherein the detectable property of the DNA sampleindicates an extent of denaturation of the DNA and the detectableproperty of the temperature reference material is correlated to thetemperature.

In some embodiments, the detectable property is measured with an opticaldetection system. In other embodiments, detectable signal from thetemperature reference material provides feedback to the heating system.In some embodiments, the DNA sample and the temperature referencematerial are alternated in the chamber. In other embodiments, whereinthe temperature reference material brackets the DNA sample in both spaceand time. In some embodiments, the detectable property emanating fromthe temperature reference materials bracketing the DNA sample ismeasured to determine a spatial temperature gradient and any temporalfluctuation.

In further embodiments, the DNA sample and the temperature referencematerial are mixed, wherein the temperature reference material has adetection signature that is discernible from that of the DNA sample andwherein data from both the sample and the temperature reference materialare collected at the same place and the same time. In some embodiments,the temperature reference material is also a flow marker. In someembodiments, the container is a microfluidic chip and the chambers aremicrofluidic channels in the chip. In some embodiments, the containercomprises at least three chambers wherein at least two of the chambersare spatially separated from each other and contain temperaturereference materials and the detectable property emanating from thetemperature reference materials is measured to determine a spatialtemperature gradient. In other embodiments, at least two chamberscontaining DNA samples are located between two chambers containing thetemperature reference material. In some embodiments, the temperaturereference material is a known DNA mixture. In other embodiments, thetemperature reference material is a thermochromatic material.

The above and other embodiments of the present invention are describedbelow with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various embodiments of the presentinvention. In the drawings, like reference numbers indicate identical orfunctionally similar elements.

FIG. 1 is a diagram illustrating a system according to an embodiment ofthe present invention.

FIG. 2 is a diagram illustrating a system according to anotherembodiment of the present invention.

FIG. 3 shows a microfluidic device that is capable of performing meltcurve data collection according to an embodiment of the presentinvention.

FIG. 4 is a diagram illustrating the spatial relationship of a DNAsample and a temperature reference material according to an embodimentof the present invention.

FIG. 5 is a diagram illustrating the spatial relationship of a DNAsample and a temperature reference material according to anotherembodiment of the present invention.

FIG. 6 is a diagram illustrating the spatial relationship of a DNAsample and a temperature reference material according to an additionalembodiment of the present invention.

FIG. 7 is a diagram illustrating the spatial relationship of a DNAsample and a temperature reference material according to a furtherembodiment of the present invention.

FIG. 8 is a diagram illustrating the spatial relationship of a DNAsample and a temperature reference material according to anotherembodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention has several embodiments and relies on patents,patent applications and other references for details known to those ofthe art. Therefore, when a patent, patent application, or otherreference is cited or repeated herein, it should be understood that itis incorporated by reference in its entirety for all purposes as well asfor the proposition that is recited.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, N.Y., Gait,Oligonucleotide Synthesis: A Practical Approach, 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rdEd., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5th Ed., W. H. Freeman Pub., New York, N.Y., all of whichare herein incorporated in their entirety by reference for all purposes.

As described above, the known systems and methods for melt curveanalysis suffer from some amount of uncertainty and lack ofreproducibility, inter alia, in terms of temperature control andmeasurement systems. The present invention provides systems and methodsfor temperature referencing for melt curve data collection. The presentinvention provides improvements in melt curve analysis technique byreducing the impact of spatial and temporal temperature fluctuations.

As shown in FIG. 1, an embodiment of the present invention provides asystem for performing melt curve data collection comprising a container101, a heating system 108 and a detection system 106. Container 101comprises at least two chambers 102 a and 102 b. In one embodiment, thechambers have the same physical dimensions. In other embodiments, thechambers are located in close proximity to one another such that theyare in close thermal connection. In some embodiments, the chambers arelocated less than 1 mm from each other. In other embodiments, thechambers are located less than 0.3 mm from each other. In someembodiments, heating system 108 supplies heat to all of the chamberssimultaneously. In some embodiments, an optional temperature sensormonitors the container and provides feedback control to the heatingsystem. In some embodiments, detection system 106 is an opticaldetection system. In other embodiments, detection system 106 monitorsthe optical properties (e.g., fluorescence emission) from the chamberssimultaneously. The system may further comprise an excitation source 104(e.g., a laser or other excitation source) for illuminating thechambers. Detection system 106 detects light (e.g., fluorescence orother light) that comes from the chambers after illumination byexcitation source 104. The system may further comprise a control unit110. Control unit 110 controls heating system 108 and may receivefeedback from the heating system. In some embodiments, a detectablesignal (e.g., fluorescence emission) received by the detection system106 provides feedback to the heating system through control unit 110. Inother embodiments, excitation source 104 provides feedback to thecontrol unit 110 for controlling detection of the detectable signal bydetection system 106.

In one embodiment, at least one chamber of the system contains a testsample of unknown DNA (i.e., DNA sample) to be tested (i.e., measured bymelt curve analysis) and at least one chamber contains a temperaturereference material. In some embodiments, the DNA sample comprises PCRamplicons. In other embodiments, the PCR amplicons contain a dsDNAbinding dye. Suitable dsDNA binding dyes included SYBR® Green 1(Invitrogen Corp., Carlsbad, Calif.), SYTO® 9 (Invitrogen Corp.,Carlsbad, Calif.), LC Green® (Idaho Technologies, Salt Lake City, Utah)and Eva Green™ (Biotium Inc, Hayward, Calif.). In further embodiments,the DNA sample comprises an unlabeled probe and single stranded ampliconproduced by asymmetric PCR. In some embodiments, the chambers contain atleast two spatially separated temperature reference materials todetermine a spatial temperature gradient. In other embodiments, thetemperature reference materials bracket the DNA sample in both space andtime. In further embodiments, the temperature reference material ismixed with the DNA sample. In this embodiment, the temperature referencematerial has a detection signature that is discernible from that of theDNA sample. In some embodiments, data from both the sample and thetemperature reference material are collected at the same place and thesame time.

In some embodiments, the container is a microfluidic chip and thechambers are microfluidic channels in the chip. In some embodiments, thecontainer comprises at least three chambers wherein at least two of thechambers are spatially separated from each other and contain atemperature reference material to determine a spatial temperaturegradient. In other embodiments, at least two chambers containing DNAsamples are located between two chambers containing the temperaturereference material.

Heating system 108 provides a substantially steadily increasing amountof heat to the chambers to cause the DNA present in the chamber(s) tomelt (i.e., to cause the dsDNA in the chamber to transition to ssDNA)for the generation of a melt curve. In one example, heating system 108may provide a thermal ramp rate of typically 0.1° C. to 2° C. persecond, with the preferred ramp rate being between 0.5° C. and 1° C. persecond. Typically, the temperature may increase from temperature t1(e.g., about 65° C.) to temperature t2 (e.g., about 95° C.). In someembodiments, heating system 108 is configured to provide heat to and/orabsorb heat from the chambers and, thus, may include one or more heatsources and/or heat sinks (e.g., heating system 108 may include apeltier device or other heat source or sink).

The temperature reference material is a known standard that exhibits awell-characterized and reproducible optical property change as afunction of temperature. In some embodiments, the temperature referencematerial is a known DNA mixture. In some embodiments, the DNA mixture isa mixture of homogeneous short DNA duplexes with a known meltingtransition. In other embodiments, the DNA mixture is a mixture of DNAduplexes having more than one melting transition. In furtherembodiments, the DNA mixture is a mixture of longer DNA duplexes withmultiple melting domains. In some embodiments, the temperature referencematerial is a thermochromatic material. In some embodiments, thethermochromatic material is a thermochromic leuco dye. In otherembodiments, the thermochromatic material is a thermochromic liquidcrystal material. Examples of thermochromic leuco dyes include, but arenote limited to, spirolactones, fluorans, spiropyrans and fulgides.Examples of thermochromic liquid crystal materials include, but are notlimited to cholesteryl nonanoate (also called cholesteryl pelargonate,3β-cholest-5-en-3-ol nonanoate or cholest-5-ene-3-β-yl nonanoate) andcyanobiphenyls.

As shown in FIG. 2, the present invention provides a system forperforming melt curve data collection comprising a container 202, aheating system 108 and a detection system 106. In this embodiment of theinvention, container 202 comprises at least one chamber that isrefillable without moving the container. Container 202 contains a DNAsample to be tested and a temperature reference material. In someembodiments, heating system 108 supplies heat to all of the chamberssimultaneously. In some embodiments, an optional temperature sensormonitors the container and provides feedback control to the heatingsystem. In some embodiments, detection system 106 is an opticaldetection system. In other embodiments, detection system 106 monitorsthe optical properties (e.g., fluorescence emission) from the chamberssimultaneously. The system may further comprise an excitation source 104(e.g., a laser or other excitation source) for illuminating thechambers. Detection system 106 detects light (e.g., fluorescence orother light) that comes from the chambers after illumination byexcitation source 104. The system may further comprise a control unit110. Control unit 110 controls heating system 108 and may receivefeedback from the heating system. In some embodiments, a detectablesignal (e.g., fluorescence emission) received by the detection system106 provides feedback to the heating system through control unit 110. Inother embodiments, excitation source 104 provides feedback to thecontrol unit 110 for controlling detection of the detectable signal bydetection system 106. Thus, this embodiment of the invention providescontainer 202 having chambers that can be emptied and refilled with newsolutions without needing to physically remove and replace the entiresample container with respect to excitation source 104, detection system106, heating system 108 and control unit 110.

In some embodiments, the DNA sample and the temperature referencematerial are alternated in the chamber within container 202. In otherembodiments, the temperature reference material brackets the DNA samplein both space and time. An illustration of the temperature referencematerial bracketing the DNA sample is shown in FIG. 2. In furtherembodiments, the DNA sample and the temperature reference material aremixed. In this embodiment, the temperature reference material has adetection signature that is discernible from that of the DNA sample. Insome embodiments, data from both the sample and the temperaturereference material are collected at the same place and the same time. Insome embodiments, the temperature reference material is also a flowmarker for monitoring flow through the chamber.

In some embodiments, the container comprises at least three chamberswherein at least two of the chambers are spatially separated from eachother and contain a temperature reference material to determine aspatial temperature gradient, such as illustrated in FIG. 2. In otherembodiments, at least two chambers containing DNA samples are locatedbetween two chambers containing the temperature reference material. Insome embodiments, the temperature reference material is a known DNAmixture, such as described herein. In other embodiments, the temperaturereference material is a thermochromatic material, such as describedherein.

In some embodiments, such as shown in FIG. 3, the container is amicrofluidic chip and the chambers are microfluidic channels in thechip. As shown in FIG. 3, chip 302 includes a number of microfluidicchannels 308. In the example shown, there are 32 microfluidic channels,but it is contemplated that chip 302 may have more or less than 32channels. As shown, a first portion of each microfluidic channel may bewithin a PCR processing zone 304 and a second portion of eachmicrofluidic channel may be within a melt analysis zone 306. As furthershown, zone 306 may immediately follow zone 304 and the length of zone304 may be significantly greater than the length of zone 306. An exampleof a microfluidic chip that can be used in connection the presentinvention is described in U.S. Patent Application Publication No.2008/0003593, incorporated herein by reference. An example of a systemand method for performing PCR in a microfluidic chip is described inU.S. Patent Application Publication No. 2008/0003588, incorporatedherein by reference. Other microfluidic chips are well known in the art.

The present invention also provides a method of performing melt curvedata collection. In accordance with one aspect, the method comprises (a)providing a container which includes at least two chambers that are inclose thermal connection, (b) introducing a DNA sample to be tested intoat least one of the chambers and a temperature reference material intoat least one of the other chambers, (c) heating the chambers from afirst temperature to a second temperature, and (d) measuring adetectable property emanating from the DNA sample and a detectableproperty emanating from the temperature reference material. Thedetectable property of the DNA sample indicates an extent of thedenaturation (i.e., melting) of the DNA in the sample. The detectableproperty of the temperature reference material is correlated to thetemperature.

In one example, the DNA sample and the temperature reference materialare as described herein. The chambers are heated in such a manner that athermal ramp rate of typically 0.1° C. to 2° C. per second, with thepreferred ramp rate being between 0.5° C. and 1° C. per second, isproduced. Typically, the temperature may increase from temperature t1(e.g., about 65° C.) to temperature t2 (e.g., about 95° C.).

In one embodiment, at least one of the chambers contains a temperaturereference material. In this embodiment, melt data from the DNA sampleand data from the temperature reference material are collectedsimultaneously as the temperature is ramped. Because the chambers aresmall and in close proximity, and the heater has been designed to supplyheat uniformly, the temperature difference between the two chambersshould be small. These two data sets are then compared to infer theactual melt properties of the DNA sample.

In some embodiments, the chambers contain at least two spatiallyseparated temperature reference materials and the detectable propertyemanating from the temperature reference materials is measured todetermine a spatial temperature gradient. In other embodiments, thetemperature reference materials bracket the DNA sample in both space andtime. In some embodiments, the detectable property emanating from thetemperature reference materials bracketing the DNA sample is measured todetermine a spatial temperature gradient and any temporal fluctuation.By having at least two spatially separated temperature referencesmeasured simultaneously, the temperature gradient can be measured andthe data compensated to account for the gradient. For example, thetemperature at the location of an unknown sample could be estimated byinterpolating the results from temperature standards that surround thatlocation.

In further embodiments, the temperature reference material is mixed withthe DNA sample. This method could be the ultimate in accuracy becausethe temperature reference is measured in the same location and at thesame time as the unknown sample. This method requires the use of amaterial whose optical property change with temperature is discerniblefrom the melt-dependent fluorescence from the DNA/binding dye undertest. There are a number of physical properties that could conceivablybe measured including absorbance and luminescence the efficiency,wavelength, polarization, or lifetime of emitted fluorescence,absorbance vs. wavelength, birefringence, etc. The addition of thetemperature standard would ideally not interfere with the DNA meltmeasurement in any way, i.e. it would not alter the melt properties,would not affect the measurement process, and also would preferably notinterfere with PCR.

In some embodiments, the detectable property is measured with an opticaldetection system as described herein. In other embodiments, detectablesignal from the temperature reference material provides feedback to theheating system. In further embodiments, the container is a microfluidicchip and the chambers are microfluidic channels in the chip. In someembodiments, the container comprises at least three chambers wherein atleast two of the chambers are spatially separated from each other andcontain temperature reference materials and the detectable propertyemanating from the temperature reference materials is measured todetermine a spatial temperature gradient. In other embodiments, at leasttwo chambers containing DNA samples are located between two chamberscontaining the temperature reference material. In some embodiments, thetemperature reference material is a known DNA mixture, such as describedherein. In other embodiments, the temperature reference material is athermochromatic material, such as described herein.

In a second aspect, the method of the present invention comprises (a)providing a container which includes at least one chamber that isrefillable without moving the container, (b) introducing a DNA sample tobe tested into at least one of the chambers and a temperature referencematerial into at least one of the chambers, (c) heating the chambersfrom a first temperature to a second temperature, and (d) measuring adetectable property emanating from the DNA sample and a detectableproperty emanating from the temperature reference material. Thedetectable property of the DNA sample indicates an extent ofdenaturation (i.e., melting) of the DNA in the sample. The detectableproperty of the temperature reference material is correlated to thetemperature.

In this aspect, new DNA samples and temperature reference material aresimply pumped into the refillable chambers. There is no need tophysically remove and replace the entire sample container with respectto the optical and temperature control systems. This attribute reducesthe chance of run-to-run temperature fluctuations because the thermalproperties of the container, as well as its contact with the heater (andadditional temperature sensors), remains the same. In addition, theimpact of temperature fluctuations is further reduced in this aspectbecause the method is capable of measuring the temperature referencestandards in the same channel, by sequentially alternating thetemperature reference materials with the unknown DNA samples. Analogousto the idea of bracketing the unknown samples in space at the same time,this idea is to bracket the unknown samples in time, at the samelocation.

In one example, the DNA sample and the temperature reference materialare as described herein. The chambers are heated in such a manner that athermal ramp rate of typically 0.1° C. to 2° C. per second, with thepreferred ramp rate being between 0.5° C. and 1° C. per second, isproduced. Typically, the temperature may increase from temperature t1(e.g., about 65° C.) to temperature t2 (e.g., about 95° C.).

In some embodiments, the detectable property is measured with an opticaldetection system as described herein. In other embodiments, detectablesignal from the temperature reference material provides feedback to theheating system. In some embodiments, the DNA sample and the temperaturereference material are alternated in the chamber. In other embodiments,wherein the temperature reference material brackets the DNA sample inboth space and time. In some embodiments, the detectable propertyemanating from the temperature reference materials bracketing the DNAsample is measured to determine a spatial temperature gradient and anytemporal fluctuation.

In further embodiments, the DNA sample and the temperature referencematerial are mixed, wherein the temperature reference material has adetection signature that is discernible from that of the DNA sample andwherein data from both the sample and the temperature reference materialare collected at the same place and the same time. In some embodiments,the temperature reference material is also a flow marker. In someembodiments, the container is a microfluidic chip and the chambers aremicrofluidic channels in the chip. In some embodiments, the containercomprises at least three chambers wherein at least two of the chambersare spatially separated from each other and contain temperaturereference materials and the detectable property emanating from thetemperature reference materials is measured to determine a spatialtemperature gradient. In other embodiments, at least two chamberscontaining DNA samples are located between two chambers containing thetemperature reference material. In some embodiments, the temperaturereference material is a known DNA mixture. In other embodiments, thetemperature reference material is a thermochromatic material.

An example of a container with refillable chambers is a microfluidicchip containing channels or a microcapillary system. New DNA sampleboluses and temperature reference material boluses are simply pumpedinto the melt detection region, without moving the container. Thisattribute reduces the chance of run-to-run temperature fluctuationsbecause the thermal properties of the container, as well as its contactwith the heater (and additional temperature sensors), remains the same.To further reduce the impact of temperature fluctuations, thisembodiment is capable of measuring the temperature reference standardsin the same channel, by sequentially alternating the reference solutionswith the unknown test samples. Analogous to the idea of bracketing theunknown samples in space at the same time, this idea is to bracket theunknown samples in time, at the same location.

Optionally, in this embodiment, the sequential temperature referencematerial boluses may serve a dual purpose. For example, if used inconjunction with a flowing real-time PCR system (see U.S. PatentApplication Publication No. 2008/0003588, incorporated herein byreference), these temperature reference material boluses could also beused as flow markers to measure the flow rate and/or dispersion along achannel.

In another embodiment, the temperature reference materials are used tobracket unknown DNA sample boluses in both space and time. In otherwords, the previous embodiments are combined and used together.

In another embodiment, the temperature references are mixed into theunknown DNA sample. This method could be the ultimate in accuracybecause the temperature reference is measured in the same location andat the same time as the unknown sample. This method requires the use ofa material whose optical property change with temperature is discerniblefrom the melt-dependent fluorescence from the DNA/binding dye undertest. There are a number of physical properties that could conceivablybe measured including absorbance and luminescence the efficiency,wavelength, polarization, or lifetime of emitted fluorescence;absorbance vs. wavelength, birefringence, etc. The addition of thetemperature standard would ideally not interfere with the DNA meltmeasurement in any way, i.e. it would not alter the melt properties,would not affect the measurement process, and also would preferably notinterfere with PCR.

In another embodiment, the optical signature from a temperaturereference material is used to provide feedback to the heater, allowingfor more accurate temperature control at that location.

Examples of the present invention (of the many embodiments and modes ofoperation) in which the container is a planar microfluidic chip with oneor more microchannels are illustrated in FIGS. 4-7. Boluses of PCRproducts (DNA sample or test boluses) to be tested by melt curveanalysis flow through these channels at a steady rate. These examplesillustrate examples in which DNA sample boluses are alternated orcombined with temperature reference material boluses in a microfluidicchannel. In each example, fluorescence signals from DNA melting orsignals from the temperature reference material are collected. Thisprocess may be repeated many times without physically moving orreplacing the microfluidic chip. Data from temperature referencematerials are used to determine spatial temperature gradients. Data fromtemperature references at different times (i.e. Melt 1 vs. Melt 2) areused to determine if temporal fluctuations have occurred.

FIG. 4 illustrates an example of the present invention in which a testbolus containing DNA to be analyzed (400 a, 400 b) alternates with atemperature reference bolus (402 a, 402 b). Each test bolus and eachtemperature reference bolus are separated by spacer fluid (404 a, 404 b,404 c, 404 d).

FIG. 5 illustrates an example of the present invention in which a testbolus containing DNA to be analyzed (500 a, 500 b, 500 c, 500 d)alternates with a bolus that contains a combined flow marker and atemperature reference material (502 a, 502 b, 502 c, 502 d, 502 e).

FIG. 6 illustrates an example of the present invention in which a testbolus contains both the DNA to be analyzed and the temperature referencematerial (600 a, 600 b, 600 c, 600 d). The test boluses are separated byspacer fluid (602 a, 602 b, 602 c, 602 d, 602 e).

FIG. 7 illustrates an example of the present invention in which themicrofluidic chip contains three channels (lanes 1-3). A test boluscontaining DNA to be analyzed (700 a, 700 b) is bracketed by bolusesthat contain a combined flow marker and temperature reference material(702 a, 702 b, 702 c, 702 d, 702 e, 702 f). The test boluses and thetemperature reference boluses in the same channel are separated byspacer fluid (704 a, 704 b, 704 c, 704 d, 704 e).

FIG. 8 illustrates an example (of the many embodiments and modes ofoperation) in which the container is a planar microfluidic chip with 32parallel microchannels (lanes 1-32). Boluses of PCR products (DNA sampleboluses) (800 a, 800 b, 800 c, 800 d) to be tested by melt curveanalysis flow through these channels at a steady rate, alternating witha spacer fluid (802 a, 802 b, 802 c, 802 d, 802 e). Some of the channels(e.g., lanes 1, 11, 22, and 32 as shown in this illustration) contain atemperature reference material (804 a, 804 b, 804 c, 804 d). As a firstgroup of DNA sample boluses reaches the melt zone, all 32 channels aresubjected to the temperature ramp (Melt 1) while fluorescence signalsfrom DNA melting and signals from the temperature reference material arecollected from all the lanes. Later, when the second group of DNA sampleboluses reaches the melt zone detector, the melt measurement isperformed again (Melt 2). This process may be repeated many times (Melt3, 4, etc.) without physically moving or replacing the microfluidicchip. Data from temperature reference materials measured simultaneouslyat different locations are used to determine spatial temperaturegradients. Data from temperature references at different times (i.e.Melt 1 vs. Melt 2) are used to determine if temporal fluctuations haveoccurred.

As evident from the above description, the present invention providesways to reduce the impact of temperature gradients and fluctuations in asystem for melt curve data collection.

By keeping the chamber in close thermal contact and with proper geometryof the heater, instantaneous temperature differences between chambers iskept small.

Using data from a temperature reference inside a chamber located near tothe sample chamber, rather than a temperature sensor external to thecontainer (which may experience significant temperature offsets due tothe dynamic nature of the temperature ramp) could provide a moreaccurate and precise estimate of the temperature in the sample chamber.

Using data from more than one temperature reference inside spatiallyseparated chambers provides information on the magnitude of temperaturegradients and may allow one to more precisely estimate the temperatureat other locations.

Using a refillable chamber (e.g, via a microcapillary tube ormicrofluidic channel) allows one to test different solutions withoutmoving the container, which could alter the properties of thetemperature control and measurement system or the optical measurementsystem. This should result in greater reproducibility from measurementto measurement.

By measuring temperature reference materials sequentially in the samechamber as the DNA test samples, one has the ability to monitor thedegree of temperature fluctuations between measurements over the courseof minutes.

By mixing a temperature reference material directly into the DNA samplebolus and measuring both simultaneously at the same location, errors dueto gradients and temporal fluctuations may be eliminated. This requiresa reference material that does not interfere with the DNA meltmeasurement or other processes.

The above methods used alone or in combination would enhance theusefulness of the melt curve analysis technique by improving theaccuracy and reliability of the temperature measurement.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. Variations of thoseembodiments may become apparent to those of ordinary skill in the artupon reading the foregoing description. The inventors expect skilledartisans to employ such variations as appropriate, and the inventorsintend for the invention to be practiced otherwise than as specificallydescribed herein. Accordingly, this invention includes all modificationsand equivalents of the subject matter recited in the claims appendedhereto as permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contradicted by context.

What is claimed is:
 1. A system for performing melt curve datacollection comprising: a container comprising at least three chambers,wherein at least two of the chambers are spatially separated from eachother, and contain a temperature reference material, and at least onechamber contains a DNA test sample; a heating system to heat thecontainer, a detection system, wherein a detectable signal from thetemperature reference material provides feedback to the heating system;and a control system for determining a spatial temperature gradient dueto temporal fluctuation within the container to calculate a temperatureat a location of the DNA test sample based on temperature results fromthe temperature reference material that surrounds the location of theDNA test sample.
 2. The system of claim 1, wherein the heating systemsupplies heat to all of the chambers simultaneously.
 3. The system ofclaim 1, wherein the chambers contain at least two spatially separatedtemperature reference materials to determine a spatial temperaturegradient.
 4. The system of claim 1, wherein the temperature referencematerial brackets the DNA test sample in both space and time.
 5. Thesystem of claim 1, wherein the temperature reference material is mixedwith the DNA test sample.
 6. The system of claim 1, wherein thedetection system is an optical detection system.
 7. The system of claim1, wherein the container is a microfluidic chip and the chambers aremicrofluidic channels in the chip.
 8. The system of claim 1, wherein atleast two chambers containing DNA test samples are located between twochambers containing the temperature reference material.
 9. The system ofclaim 1, wherein the temperature reference material is a known DNAmixture.
 10. The system of claim 1, wherein the temperature referencematerial is a thermochromatic material.
 11. A system for performing meltcurve data collection, said system comprising: a container comprising atleast three chambers that are refillable without moving the container,wherein at least two of the chambers are spatially separated from eachother and contain a temperature reference material, and at least onechamber contains a DNA test sample; a heating system to heat thecontainer; a detection system, wherein a detectable signal from thetemperature reference material provides feedback to the heating system;a control system for determining a spatial temperature gradient due totemporal fluctuation within the container to calculate a temperature ata location of the DNA test sample based on temperature results from thetemperature reference material that surrounds the location of the DNAtest sample.
 12. The system of claim 11, wherein the heating systemsupplies heat to all of the at least three chambers simultaneously. 13.The system of claim 11, wherein the detection system is an opticaldetection system.
 14. The system of claim 11, wherein the DNA testsample and the temperature reference material are alternated in thechamber.
 15. The system of claim 11, wherein the temperature referencematerial brackets the DNA test sample in both space and time.
 16. Thesystem of claim 11, wherein the DNA test sample and the temperaturereference material are mixed, wherein the temperature reference materialhas a detection signature that is discernible from that of the DNA testsample, and wherein data from both the sample and the temperaturereference material are collected at the same place and the same time.17. The system of claim 11, wherein the temperature reference materialis also a flow marker.
 18. The system of claim 11, wherein at least twochambers containing DNA test samples are located between two chamberscontaining the temperature reference material.
 19. The system of claim11, wherein the temperature reference material is a known DNA mixture.20. The system of claim 11, wherein the temperature reference materialis a thermochromatic material.
 21. The system of claim 1, wherein atleast two of the chambers are located less than 1 mm from each other.22. The system of claim 1, wherein the detection system monitors opticalproperties from the chambers simultaneously.
 23. The system of claim 11,wherein at least two of the chambers are located less than 1 mm fromeach other.
 24. The system of claim 11, wherein the detection systemmonitors optical properties from the chambers simultaneously.